Mouse anti human ctnt

For flow cytometric analyses, EBs were dissociated overnight in 1 mg/ml collagenase B (Roche) at 37°C, followed by incubation in TrypLE (Invitrogen) the next morning for 10–15 min to break up remaining EBs. To stain total cardiomyocytes, cells were stained with 1∶500 anti-human SIRPα-PE/Cy7 (BioLegend) and 1∶250 anti-human CD90-FITC (BD Pharmingen) for 1 h at 4°C in PBS/10% FBS staining buffer. Cells were filtered through a 40-µm cell strainer (Fisher) and resuspended at 10⁶ cells/mL in staining buffer for cell sorting. Sorting was performed on an AriaII cell sorter (BD Biosciences). Flow cytometric gates were set using control cells stained with the appropriate isotype control antibody. To determine cardiomyocyte purity, dissociated single cells were fixed with 4% PFA for 15 min at room temperature. Cells were then blocked in 2% BSA, 2% FBS, and 0.01% Triton for 1 h at room temperature. The primary antibody mouse-anti-human cTNT (ThermoScientific, clone 13–11) was conjugated to AlexaFluor 488 invitro using the Zenon Mouse IgG Labeling Kit (Invitrogen), according to manufacturer’s instructions. Conjugated primary antibody was added to blocking solution at 1∶100 final dilution of cTNT antibody for 2 h at room temperature. Cells were analyzed on an LSR-II (BD Biosciences). Data were analyzed using FlowJo software, Version 9.3.2.